A common and useful technique in the biological and physical sciences involves separation or isolation of compounds of discrete density by density fractionation. One method to effect this separation is density gradient centrifugation. In the biological sciences, variations in cell types, differences in the phenotype of cellular components, variation in DNA base or isotope composition, and isolation of the viral genomes and proteins have all been effected by density gradient centrifugation. To effect this technique, a solution of continuously varying concentrations is contained within a centrifuge tube. The solution will be most concentrated at the bottom of the tube and least concentrated at the top. A sample to be analyzed is then introduced into the tube. Next, a centrifugal field is applied to the tube at the top. As the centrifugal force is applied, the compounds within the tube will migrate toward the outer radius (i.e. tube bottom for swinging bucket rotors, tube side for vertical tube rotors) based on density and separate into isopycnic bands at discrete levels within the centrifuge tube. These bands can then be recovered from the tube, further purified if necessary, and analyzed.
To effect a density gradient centrifugal separation, a density gradient must be formed. This requires layering solutions of lighter density on top of solutions of heavier density. Solutions of sucrose of decreasing concentrations can be layered one on top of another to form a linear gradient from, for example 10-40% from the top of the tube to the bottom. In a linear gradient, the density varies equally along the length of the tube. The solutions of higher density are more viscous. Thus, a density gradient establishes a viscosity gradient in the tube as well. Ordinarily, for swinging bucket rotors, the g forces are greatest at the bottom of the tube which has a greater radius from the center of rotation than the top of the tube. The viscosity gradient counteracts the g force difference, allowing a nearly uniform migration rate of particles throughout the tube. The gradient also prevents mixing of the contents of the tube during acceleration and deceleration. This allows the formation of discrete isopycnic bands.
Several techniques have been developed to form linear density gradients. A first technique involves having a sucrose solution at a uniform concentration undergo repeated cycles of freezing and thawing. This freeze-thaw technique produces a gradient because the ice floats, excluding the sugar. However, this method has several drawbacks. The technique has poor reproducibility due to variations in the freeze-thaw cycles and is time intensive. Furthermore, any other compounds in solution in addition to the sugar would also be excluded from forming ice crystals, resulting in changes to the buffer concentration throughout the tube. This could affect the stability of a sample the researcher is seeking to separate.
A second technique of forming sucrose density gradients entails simply layering various concentrations of sucrose by hand. In this technique, a plurality of sucrose solutions are each layered into a centrifuge tube. The solution with the lowest concentration would be added first to the tube, with subsequent layers added by pipette from the bottom of the tube, causing flotation of previously inserted layers. This method will not result in a complete gradient, but instead will be stepwise concentration changes. This method is a laborious process with low reproducibility.
In U.S. Pat. No. 5,171,539, the process of gradient formation is automated to allow greater reproducibility. This patent describes an apparatus for holding centrifuge tubes and moving the tubes into a programmed orientation. By including a low concentration sucrose solution floated over a high concentration sucrose solution, the rotation of the tube would produce a mechanically reproducible gradient. The patent notes at col. 5, lines 46-49, that if sucrose is not used to make the gradient, the gradient could alternatively be formed by using Percoll. This technique, although improving the reproducibility, requires purchase of additional gradient forming apparatus and required time for the apparatus to form the density gradient.
Some ability to produce self-forming gradients is known. For example, U.S. Pat. No. 4,480,038 describes using a 60% Percoll gradient to separate cells. A very small quantity of the sugar sucrose is added for osmolarity. Under a relative short spin time (45 min.) under a centrifugal field of 100,000.times.g density markers would separate. However, many experimental protocols for separating compounds with low migration rates would require density gradients that are stable for spin times of several hours. For example, subcellular particles, unlike the cells described in U.S. Pat. No. 4,480,038, have low sedimentation rates. If a more stable self-forming density gradient could be developed, it would greatly simplify and improve density gradient separations that require longer spin times.
It is the object of this invention to provide a process for producing a self-forming density gradient. Because this gradient would be self-forming, producing the gradient would be rather simple and would take minimal time. Additionally, a self-forming gradient would not require the purchasing of additional instrumentation since the gradient would automatically form in a centrifugal field.
Another object of the invention is to devise a method for enhancing the reproducibility of gradient formation.
Another object of the invention is to devise a density gradient that can be used in high capacity rotors allowing for greater experimental throughput.
Finally, the invention should require only inexpensive materials, be easy to prepare, easy to use, and adaptable to a variety of experimental protocols.